The long-term goal of our research has been to understand, on a molecular level, how cytoplasmic protein- tyrosine kinases regulate the growth properties of immune cells in reponse to extracellular stimuli. We have focused our studies on the tyrosine kinases involved in the activation of B lymphocytes with a special emphasis on the tyrosine kinase, Syk, which was discovered as part of this work. We hypothesize that Syk plays a critical regulatory role in determining the ultimate physiological outcome of antigen/antigen receptor interactions through the differential phosphorylation of its tyrosine residues. Of particular interest is a multi- functional docking site that differentially binds positive effectors depending on the identity and stoichiometry of phosphorylation of a pair of closely spaced tyrosines. While Syk has been best characterized as an essential component of the antigen receptor-signaling machinery, increasing evidence indicates that it also plays fundamental roles in the responses of immune cells both to tumor necrosis factor (TNF)-family receptors and external stress stimuli. We hypothesize that these additional roles for Syk are dependent both on the location of the kinase within the cell and the proteins and substrates with which it interacts. To explore these questions, we plan to accomplish three specific aims: 1) to investigate the role of the multi-functional docking site on Syk on coupling the B cell receptor for antigen to downstream signal transduction pathways, 2) to investigate the role of Syk in regulating signal transduction pathways downstream of TNF-family receptors, and 3) to investigate the role of Syk in the modulation of cellular responses to stress stimuli through the identification and characterization of its binding partners and substrates. Methodologies to be employed include 1) genetic, biochemical and microscopic evaluations of protein-protein interactions and their functions, 2) structure determination of protein-protein interactions by high resolution NMR, and 3) proteomic analysis of interacting proteins and kinase substrates.